Tamily A. Weissman

The embryonic ventricular zone (VZ) of the cerebral cortex contains migrating neurons, radial glial cells, and a large population of cycling progenitor cells that generate newborn neurons. The latter two cell classes have been assumed for some time to be distinct in both function and anatomy, but the cellular anatomy of the progenitor cell type has remained poorly defined. Several recent reports have raised doubts about the distinction between radial glial and precursor cells by demonstrating that radial glial cells are themselves neuronal progenitor cells (Malatesta et al., 2000; Hartfuss et al., 2001; Miyata et al., 2001; Noctor et al., 2001). This discovery raises the possibility that radial glia and the population of VZ progenitor cells may be one anatomical and functional cell class. Such a hypothesis predicts that throughout neurogenesis almost all mitotically active VZ cells and a substantial percentage of VZ cells overall are radial glia. We have therefore used various anatomical, immunohistochemical, and electrophysiological techniques to test these predictions. Our data demonstrate that the majority of VZ cells, and nearly all mitotically active VZ cells during neurogenesis, both have radial glial morphology and express radial glial markers. In addition, intracellular dye filling of electrophysiologically characterized progenitor cells in the VZ demonstrates that these cells have the morphology of radial glia. Because the vast majority cycling cells in the cortical VZ have characteristics of radial glia, the radial glial precursor cell may be responsible for both the production of newborn neurons and the guidance of daughter neurons to their destinations in the developing cortex.

Advances in imaging and cell-labeling techniques have greatly enhanced our understanding of developmental and neurobiological processes. Among vertebrates, zebrafish is uniquely suited for in vivo imaging owing to its small size and optical translucency. However, distinguishing and following cells over extended time periods remains difficult. Previous studies have demonstrated that Cre recombinase-mediated recombination can lead to combinatorial expression of spectrally distinct fluorescent proteins (RFP, YFP and CFP) in neighboring cells, creating a 'Brainbow' of colors. The random combination of fluorescent proteins provides a way to distinguish adjacent cells, visualize cellular interactions and perform lineage analyses. Here, we describe Zebrabow (Zebrafish Brainbow) tools for in vivo multicolor imaging in zebrafish. First, we show that the broadly expressed ubi:Zebrabow line provides diverse color profiles that can be optimized by modulating Cre activity. Second, we find that colors are inherited equally among daughter cells and remain stable throughout embryonic and larval stages. Third, we show that UAS:Zebrabow lines can be used in combination with Gal4 to generate broad or tissue-specific expression patterns and facilitate tracing of axonal processes. Fourth, we demonstrate that Zebrabow can be used for long-term lineage analysis. Using the cornea as a model system, we provide evidence that embryonic corneal epithelial clones are replaced by large, wedge-shaped clones formed by centripetal expansion of cells from the peripheral cornea. The Zebrabow tool set presented here provides a resource for next-generation color-based anatomical and lineage analyses in zebrafish.